Antibodies are powerful components of the immune system of many animals and especially humans. Recent advances in recombinant technology have allowed for the production of antibodies against virtually any target, for example, cancer cells, bacteria, and viruses. Typically, an antibody is produced using a cell line that has been engineered to express the antibody at high levels. The engineered cell line is subsequently grown in a culture that comprises a complex mixture of sugars, amino acids, and growth factors, as well as various proteins, including for example, serum proteins. However, separation of complete antibodies from cell by-products and culture components to a purity sufficient for use in research or as therapeutics poses a formidable challenge. The purification of the antibody molecules is especially critical if the antibodies are to be used as a drug for administration to humans.
Traditional antibody purification schemes (or trains) often comprise a chromatography step which exploits an ability of the antibody molecule to preferentially bind or be retained by the solid phase (or functionalized solid phase) of a chromatography column compared to the binding or retention of various impurities. Schemes have been proposed or carried out to purify antibodies which first bind CH2/CH3 region-containing proteins to Protein A immobilized on a solid phase, followed by removal of impurities bound to the solid phase by washing the solid phase with a hydrophobic electrolyte solvent and the subsequent recovery of the CH2/CH3 region-containing proteins from the solid phase. However, these schemes are limited in that the conditions used to preferentially bind the CH2/CH3 region-containing proteins also support binding of impurities (e.g., antibodies with incomplete CH2/CH3 regions). In the development of human therapeutics, such impurities are highly undesirable.
Accordingly, a need exists for improvements in the purification of proteins or polypeptides having constant regions, in particular, proteins having Fc regions (e.g., antibodies), produced in cell culture.